色谱2025，Vol.43Issue(2)：148-154,7.DOI:10.3724/SP.J.1123.2024.02004
超高效液相色谱-串联质谱法测定尿液中血栓素A2的3种代谢物和8-异前列腺素F2α
Determination of three metabolites of thromboxane A2 and 8-iso-prostaglandin F2a in urine by ultra performance liquid chromatography-tandem mass spectrometry
摘要
Abstract
Thromboxane A2(TXA2),a prothrombotic factor that induces platelet aggregation and thrombosis,acts as a vasoconstrictor by activating TXA2 receptors(TP receptors).TXA2 is extremely unstable and metabolizes into three major metabolites:2,3-dinor thromboxane B2(2,3-dinor-TXB2),11-dehydro TXB2(11-dh-TXB2),and 11-dehydro-2,3-dinor TXB2(11-dh-2,3-dinor-TXB2).8-Iso-prostaglandin F2α(8-iso-PGF2α),a prostaglandin-like compound widely considered the best biomarker of oxidative stress,can also activate TP receptors.The accurate quantification of TXA2 metabolites and 8-iso-PGF2α is critical in cardiovascular disease(CVD). In this study,a method based on ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)was developed for the simultaneous determination of 2,3-dinor-TXB2,ll-dh-2,3-dinor-TXB2,11-dh-TXB2,and 8-iso-PGF2α in human urine.Urine samples were collected from healthy volunteers and patients with CT-or MRI-confirmed ischemic stroke oc-curring less than 48 h earlier,and cryopreserved at-80 ℃ within 1 h after collection.The urine samples were thawed at room temperature and acidified to pH 2.0-4.0 using hydrochloric acid.The supernatant was collected after centrifugation.A total of 1 mL of each urine sample was added with 100 μL of the internal standard working solution and mixed well.The samples were loaded onto a C18 SPE column(50 mg).The SPE cartridges were preconditioned with 500 μL of methanol and then equilibrated with 500 μL of water.After sample loading,the SPE cartrid-ges were washed with 500 μL of water,500 μL of 5％methanol aqueous solution containing 0.5％(v/v)ammonia,and 500 μL of 5％methanol aqueous solution containing 2％(v/v)for-mic acid.The cartridges were dried,and the analytes were eluted with 400 μL of methanol.The eluents were dried and subsequently reconstituted with 50 μL of 13％acetonitrile aqueous solu-tion.After filtration through a filter membrane,the samples were analyzed on an ACQUITY UP-LC® BEH phenyl column(50 mmx2.1 mm,1.7 μm)via gradient elution using 2 mmol/L am-monium acetate aqueous solution containing 0.002％(v/v)ammonia and acetonitrile as the mobile phases.The flow rate was 0.3 mL/min,and the column temperature was 40 ℃.The an-alytes were determined in negative electrospray ionization and multiple-reaction monitoring modes.The four target compounds showed satisfactory linearity within the relevant ranges,with linear correlation coefficients(R2)greater than 0.99.The limits of detection of the method were 0.02 ng/mL for 2,3-dinor-TXB2 and 0.01 ng/mL for 11-dh-2,3-dinor-TXB2,11-dh-TXB2,and 8-iso-PGF2α.The limits of quantification were 0.1 ng/mL for 2,3-dinor-TXB2 and 0.05 ng/mL for 11-dh-2,3-dinor-TXB2,11-dh-TXB2,and 8-iso-PGF2α.In actual urine,the recovery rates at the LOQ level were in the range of 91.48％-104.87％.The recovery rates at low,medi-um,and high levels were in the range of 92.95％-104.90％.The intra-and inter-day precisions were in the range of 2.79％-13.01％and 4.45％-13.67％,respectively.The relative error(RE)between the average peak area of the mixed matrix and the sum of the ratios of the pure solu-tion and urine matrices was within±20％.The samples were stable at 4 ℃ for 24 h and at-70 ℃for 10 d.The developed method is the first to realize the simultaneous determination of 2,3-di-nor-TXB2,11-dh-2,3-dinor-TXB2,11-dh-TXB2,and 8-iso-PGF2α in urine.The method was used to determine the concentrations of 2,3-dinor-TXB2,11-dh-2,3-dinor-TXB2,11-dh-TXB2,and 8-iso-PGF2α in healthy controls and patients with ischemic stroke,and the results were corrected using creatinine.Binary logistic regression analysis was used to construct the prediction model,and a receiver operating characteristic(ROC)curve was drawn to evaluate the clinical diagnos-tic ability of the method for the target compounds.TXA2 was calculated as the sum of its three metabolites.The area under the curve(AUC)of TXA2 was 0.849,and the method sensitivity and specificity were 69.2％and 92.3％,respectively.The AUC of 8-iso-PGF2α was 0.775,and the method sensitivity and specificity were 84.6％and 76.9％,respectively.The proposed meth-od has good clinical value and is expected to assist in the early screening and diagnosis of is-chemic stroke.关键词
超高效液相色谱-串联质谱/2,3-地诺血栓素B2/11-脱氢-2,3-地诺血栓素B2/11-脱氢血栓素B2/8-异前列腺素F2α/尿液/缺血性脑卒中分类
化学化工引用本文复制引用
刘思佳,赵福荣,张亚莲,孙晓宇,张萌萌,侯琨,曹云峰..超高效液相色谱-串联质谱法测定尿液中血栓素A2的3种代谢物和8-异前列腺素F2α[J].色谱,2025,43(2):148-154,7.基金项目
上海市生物医药技术研究院省部级重点实验室创新能力专项资助(CX2023-04) （CX2023-04）
大连市科技人才创新支持政策计划"高层次人才团队"项目(2022RG11).Innovation Promotion Program of NHC and Shanghai Key Labs SIBPT(No.CX2023-04) （2022RG11）
Dalian Science and Technology Talent Innovation Support Policy Plan"High-Level Talent Team"Project(No.2022RG11). （No.2022RG11）
